Method for improving heat stability of RNA

ABSTRACT

A method for preparing a CDNA from a mRNA using a reverse transcriptase wherein reverse transcription is performed at a temperature at which the mRNA does not take a secondary structure, for example, at a temperature of 45° C. or more. The method is performed, for example, using a heat-labile reverse transcriptase in the presence of a substance exhibiting chaperone function having chaperone function such as saccharides. The method is performed, for example, in the presence of metal ions necessary for activation of the reverse transcriptase and a chelating agent for the metal ions such as a deoxynucleotide triphosphate. The method is capable of reverse transcription over the full length of mRNA template even if the mRNA is a long chain mRNA and, as a result, producing a full length cDNA.

This application is a divisional, of application No. 09/414,531, filedOct. 8, 1999, now U.S. Pat. No. 6,221,599, which is a divisional of Ser.No. 08/899,392, filed Jul. 23, 1997 now U.S. Pat. No. 6,013,488.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for reverse transcriptionwhich can produce a full length cDNA from a mRNA. In addition, thepresent invention relates to a method for improving heat stability ofRNA.

2. Related Art

It is known that cDNAs can be obtained from mRNAs in vitro using areverse transcriptase (RNA-dependent DNA polymerase). A projectelucidating whole human gene sequences is moving on and, in thatproject, mRNA strands are produced by using genes as templates and fulllength cDNA strands are produced in turn by using the mRNA strands astemplates. That is, synthesis of first chains of cDNA from mRNA strandsis used as a first step of production of cDNA libraries, RT-PCR and thelike.

Reverse transcription is utilized in order to obtain full length cDNAstrands from the mRNAs as described above. However, conventional reversetranscription can not afford full length cDNAs from mRNAs because theconventional reverse transcription method could not complete reversetranscription to the most end cap site of mRNAs.

According to the present inventor's examination, it was found that thefailure of complete reverse transcription is caused as follows. That is,a long chain mRNA may form a secondary structure like secondarystructure of protein and the elongation by reverse transcriptase issterically hindered at the site forming the secondary structure. As aresult, reverse transcription was not completed to the end of mRNA.

That is, current techniques for reverse transcription have a technicallimitation that the reaction is ended prematurely because of a stablesecondary structure of mRNA and thus the probability of completetranscription over the whole transcription unit including its 5′ end isextremely low. This technical limitation affects the quality oflibraries. That is, most of cloned cDNAs synthesized from the poly A atthe 3′ end using an oligo dT as a primer have only the 3′ end and do nothave the full length because of the premature termination of thesynthesis. Several attempts have been made to overcome this problem. Forexample, it was proposed that the mRNAs are pre-treated at 70° C. tounfold the secondary structure before the synthesis of the first chains.It is also possible to treat the mRNAs with methylmercury hydroxideinstead of the heat treatment. Though these techniques are effective forincreasing efficiency of the synthesis of the first chain to someextent, they are not yet sufficient to efficiently obtain full lengthcDNAs. In particular, they show particularly low efficiency for thereverse transcription of long mRNAs of several kbp or more.

Therefore, the first object of the present invention is to provide amethod capable of reverse transcription of mRNA over the full length andhence capable of providing a full length cDNA even if a long chain mRNAis used as a template.

In this respect, the present inventor has found that the above firstobject of the present invention can be achieved by performing reversetranscription at a temperature at which mRNA does not form a secondarystructure. Though the temperature range where mRNAs do not form asecondary structure may change depending on buffer composition and thelike, it is for example a range of 45° C. or more, especially, 60° C. ormore.

In such a temperature range, mRNAs can be maintained in a condition thatit does not take the secondary structure and the synthesis of the firstchain can be effected efficiently. However, it was also found that, insuch a temperature range as mentioned above, (1) the reversetranscriptase may be disadvantageously inactivated depending on the kindof the enzyme, and (2) stability of mRNA may be disadvantageouslydeteriorated (mRNA is fragmented) when metal ions necessary foractivation of reverse transcriptase such as magnesium ions and a bufferagent such as Tris [Tris(hydroxymethyl)aminomethane] are presentsimultaneously.

Therefore, the second object of the present invention is to provide amethod which is capable of reverse transcription of mRNA over the fulllength of the mRNA even if a long chain mRNA is used as a template byperforming the reverse transcription of mRNA at a temperature at whichthe mRNA does not form the secondary structure and, in addition, whichcan prevent inactivation of the enzyme by heat, i.e., activate it at anelevated temperature even when a heat-labile reverse transcriptase isused and, as a result, provide a full length cDNA with high reliability.

The third object of the present invention is to provide a method whichis capable of reverse transcription of mRNA over the full length of mRNAeven if a long chain mRNA is used as a template by performing thereverse transcription of mRNA at a temperature at which the mRNA doesnot form the secondary structure and, in addition, which can provide afull length cDNA with high reliability by using a heat-resistant reversetranscriptase.

The fourth object of the present invention is to provide a method whichis capable of reverse transcription of mRNA over the full length of mRNAeven if a long chain mRNA is used as a template by performing thereverse transcription of mRNA at a temperature at which the mRNA doesnot form the secondary structure and, in addition, which can maintainstability of mRNA and hence provide a full length cDNA with highreliability even when metal ions necessary for activation of reversetranscriptase is present, in particular, when a buffer agent such asTris is further present simultaneously.

The fifth object of the present invention is to provide a method improveheat stability of mRNA even when metal ions necessary for activation ofreverse transcriptase is present, in particular, when a buffer agentsuch as Tris is further present simultaneously.

SUMMARY OF THE INVENTION

As the first embodiment of the present invention, which can achieve theabove first object of the present invention, there is provided a methodfor preparing a cDNA from a mRNA using a reverse transcriptase whereinreverse transcription is performed at a temperature at which temperaturethe mRNA does not take a secondary structure.

As the second embodiment of the present invention, which can achieve theabove second object of the present invention, there is provided a methodfor preparing a cDNA from a mRNA using a reverse transcriptase whereinreverse transcription is performed at a temperature at which the mRNAdoes not take a secondary structure using a heat-labile reversetranscriptase in the presence of a substance exhibiting chaperonefunction.

As the third embodiment of the present invention, which can achieve theabove third object of the present invention, there is provided a methodfor preparing a cDNA from a mRNA using a reverse transcriptase whereinreverse transcription is performed at a temperature at which the mRNAdoes not take a secondary structure using a heat-resistant reversetranscriptase.

As the fourth embodiment of the present invention, which can achieve theabove fourth object of the present invention, there is provided a methodfor preparing a cDNA from a mRNA using a reverse transcriptase whereinreverse transcription is performed at a temperature at which the mRNAdoes not take a secondary structure in the presence of metal ionsnecessary for activation of reverse transcriptase, a Tris buffer and achelating agent for the metal ions.

As the fifth embodiment of the present invention, which can achieve theabove fifth object of the present invention, there is provided a methodfor improving heat stability of RNAs in a solution containing metal ionswherein the solution further contains a chelating agent for the metalions.

One of the preferred embodiments of the invention is a method forpreparing a cDNA from a mRNA using a reverse transcriptase wherein:

(1) the reverse transcription is performed at a temperature at which themRNA does not take a secondary structure,

(2) the reverse transcription is performed using a heat-labile reversetranscriptase in the presence of one or more substances exhibitingchaperone function, and

(3) the reverse transcription is performed in the presence of metal ionsnecessary for activation of the reverse transcriptase and a chelatingagent for the metal ions.

Another preferred embodiment of the invention is a method for preparinga cDNA from a mRNA using a reverse transcriptase wherein:

(1) the reverse transcription is performed at a temperature at which themRNA does not take a secondary structure,

(2) the reverse transcription is performed using a heat-labile reversetranscriptase in the presence of one or more substances exhibitingchaperone function and one or more polyalcohols, and

(3) the reverse transcription is performed in the presence of metal ionsnecessary for activation of the reverse transcriptase and a chelatingagent for the metal ions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing the results of agarose gelelectrophoresis obtained in Example 1.

FIG. 2 is a photograph showing the results of agarose gelelectrophoresis obtained in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

The first embodiment of the method for preparing a cDNA from a mRNAusing a reverse transcriptase according to the present invention ischaracterized in that the reverse transcription is performed at atemperature at which the mRNA does not take a secondary structure. The“temperature at which the mRNA does not take a secondary structure”means, for example, a temperature of 45° C. or more, more precisely, atemperature in the range of 45-90° C. As the temperature becomes higher,it becomes easier to keep the mRNA not taking a secondary structure, butthe activity of reverse transcriptase and the stability of the mRNA tendto be deteriorated. Therefore, the temperature is preferably in therange of 50-75° C.

The chain length of the mRNA used for the method of the presentinvention is not particularly limited. However, it is consideredunnecessary to use the present invention for a short chain mRNA whichdoes not take a secondary structure, whereas it is difficult to obtainreverse transcription producing a full length cDNA as to a mRNA of 4 kbpor more, in particular, 7 kbp or more. Therefore, from this point ofview, the method of the present invention is particularly useful for thereverse transcription of a mRNA of 4 kbp ore more, in particular, 7 kbpor more. However, a mRNA of less than 4 kbp is not excluded from theobjective of the present invention.

The second embodiment of the method for preparing a CDNA from a mRNAusing a reverse transcriptase according to the present invention ischaracterized in that it uses a heat-labile reverse transcriptase andthe reverse transcription is performed in the presence of a substanceexhibiting chaperone function.

In the present invention, the heat-labile reverse transcriptase means areverse transcriptase exhibiting an optimum temperature of 45° C. orlower. Examples of such a heat-labile reverse transcriptase includeSuperscript II, AMV reverse transcriptase, MuLV reverse transcriptaseand the like, but it is not limited to these.

A reverse transcriptase usually used at an ordinary temperature such asSuperscript II exhibits a lower activity at a temperature of 45° C. ormore compared to the activity at the optimum temperature and exhibitssubstantially no activity at a temperature higher than a certain level.Further, if such a reverse transcriptase is maintained at a temperatureof 50° C. or higher for a certain period of time, it no longer exhibitsthe activity even though it is returned to room temperature.

In particular, when the chain length of mRNA is long, the reversetranscription is likely to prematurely terminate before a complete cDNAis synthesized because of inactivation of the enzyme by heat and hencefull length transcription becomes difficult. Therefore, according to thepresent invention, a substance exhibiting chaperone function is added tothe reverse transcription system so that the activity of the reversetranscriptase can be maintained even at an elevated temperature (it ispossible to prevent reduction of the activity and inactivation by heat).

Examples of the substance exhibiting chaperone function includesaccharides, amino acids, polyalcohols and their derivatives, andchaperone proteins. However, the substance is not limited to these. The“chaperone function” means a function for renaturing proteins denaturedby stress such as heat shock, or a function for preventing completedenaturation of proteins by heat to maintain the native structure.

Examples of the saccharide exhibiting the chaperone function includeoligosaccharides and monosaccharides such as trehalose, maltose,glucose, sucrose, lactose, xylobiose, agarobiose, cellobiose,levanbiose, quitobiose, 2-β-glucuronosylglucuronic acid, allose,altrose, galactose, gulose, idose, mannose, talose, sorbitol, levulose,xylitol and arabitol. However, the saccharide is not limited to these.Those saccharides mentioned above can be used alone or in anycombination thereof. Among these, trehalose, sorbitol, xylitol, levuloseand arabitol exhibit strong chaperone function and marked effect foractivating enzymes at an elevated temperature.

Examples of the amino acids and derivatives thereof includeN^(e)-acetyl-β-lysine, alanine, γ-aminobutyric acid, betain, N^(α)-carbamoyl-L-glutamine 1-amide, choline, dimethylthetine, ecotine(1,4,5,6-tetrahydro-2-methyl-4-pirymidine carboxilic acid), glutamate,β-glutammine, glycine, octopine, proline, sarcosine, taurine andtrymethylamine N-oxide (TMAO). However, the amino acids and derivativesthereof are not limited to these. Those amino acids mentioned above canbe used alone or in any combination thereof. Among these, betain andsarcosine exhibit strong chaperone function and marked effect foractivating enzymes at an elevated temperature.

The substance exhibiting chaperone function include polyalcohols. Thesaccharides are included in polyalcohols and other examples of thepolyalcohols include glycerol, ethylene glycol, polyethylene glycol andthe like. Those polyalcohols can be used alone or in any combinationthereof.

The substance exhibiting chaperone function include chaperone proteins.Examples of the chaperone proteins include chaperone proteins ofThermophiric bacteria and heat shock proteins such as HSP 90, HSP 70 andHSP 60. Those chaperone proteins can be used alone or in any combinationthereof.

These substances exhibiting chaperone function show different optimumconcentrations for stabilizing the enzyme depending on the kind of theenzyme and the optimum concentration may vary among the substances forthe same enzyme. Therefore, a concentration of particular substance tobe added to a specific reaction system may be suitably decided dependingon the kinds of the substance and the enzyme such as reversetranscriptase.

To enhance the effect of the substances exhibiting chaperone functionsuch as saccharides, amino acids or chaperone proteins, one or morekinds of polyalcohols may be used in addition to one ore more kinds ofthe above substances. Examples of the polyalcohol include glycerol,ethylene glycol, polyethylene glycol and the like.

The third embodiment of the method for preparing a cDNA from a mRNAusing a reverse transcriptase according to the present invention ischaracterized in that it is carried out by using a heat-resistantreverse transcriptase.

In the present invention, a heat-resistant reverse transcriptase refersto a reverse transcriptase having an optimum temperature of about 40° C.or more. Examples of such a heat-resistant reverse transcriptase includeTth polymerase, but the heat-resistant reverse transcriptase is notlimited to this.

Tth polymerase shows an optimum temperature of 70° C. and can catalyzethe reverse transcription with a high activity in the above temperaturerange of 45° C. or higher.

The fourth embodiment of the method for preparing a cDNA from a mRNAusing a reverse transcriptase according to the present invention ischaracterized in that, when the reverse transcription is performed inthe presence of the metal ions necessary for activating the reversetranscriptase, a chelating agent for the metal ions is usedsimultaneously.

Enzymes may require metal ions for their activation. For example,Superscript II, which is a reverse transcriptase, requires magnesiumions for its activation. However, in a buffer containing magnesium ionssuch as a Tris buffer, fragmentation of mRNAs may proceed under thetemperature condition mentioned above and hence it is difficult toobtain full length cDNAs. Likewise, Tth polymerase requires manganeseions as metal ions for its activation. However, also in a buffercontaining manganese ions such as a Tris buffer, fragmentation of mRNAmay actively proceed under the temperature condition as mentioned aboveand hence it is difficult to obtain full length cDNAs.

To solve this problem, according to the method of the present invention,a chelating agent for metal ions is added to the system so that theactivity of reverse transcriptase should be maintained and thefragmentation of mRNAs can be prevented. However, if all of the metalions necessary for the activation of the reverse transcriptase arechelated, the reverse transcriptase loses its activity. Therefore, it issuitable to use a chelating agent of comparatively weak chelating power.

Examples of such a chelating agent of comparatively weak chelating powerinclude deoxynucleotide triphosphates (dNTPs). The chelating agent ofcomparatively weak chelating power is suitably used in an approximatelyequimolar amount of the metal ion. When a deoxynucleotide triphosphateis used as the chelating agent, for example, it is suitable to add anapproximately equimolar amount of deoxynucleotide triphosphate as to themetal ion. Accordingly, the amount of the chelating agent can besuitably decided with consideration to the chelating power as to theobjective metal ion, so that the reverse transcriptase activity can bemaintained and the fragmentation of mRNAs can be prevented. Thedeoxynucleotide triphosphates, dATP, dGTP, dCTP and dTTP, may be usedalone or in any combination thereof. All of the four kinds of dNTPs,dATP, dGTP, dCTP and dTTP, may be used together. Since these can servealso as substrates of the reverse transcription, all of them are usuallyused together.

A preferred, but non-limitative embodiment of the method for preparing acDNA from a mRNA using reverse transcriptase according to the presentinvention is a method characterized in that:

(1) the reverse transcription is performed at a temperature at which themRNA does not take a secondary structure, for example, a temperature of45 to 90° C., particularly preferably a temperature of around 60° C.,

(2) the reverse transcription is performed in the presence of one ormore substances exhibiting chaperone function and one or morepolyalcohols, and

(3) the reverse transcription is performed in the presence of metal ionsnecessary for activation of the reverse transcriptase and a chelatingagent for the metal ions.

For example, the method is performed by using Seperscript II as thereverse transcriptase in a Tris buffer containing deoxynucleotidetriphosphates as the chelating agents and magnesium ions.

The fifth embodiment of the present invention which is a method forimproving heat stability of RNAs in a solution containing metal ions ischaracterized in that the solution further contains a chelating agentfor the metal ions.

As mentioned above, enzymes may require metal ions for their activationand in a Tris buffer containing metal ions such as magnesium ions,fragmentation of mRNAs may proceed under an elevated temperature. In thefifth embodiment of the present invention, a chelating agent for themetal ions is added to a solution containing RNAs for improvement ofheat stability.

A chelating agent for metal ions is added to the solution so that thefragmentation of mRNAs can be prevented and if reverse transcriptasecoexists, the activity of reverse transcriptase should also bemaintained. However, if all of the metal ions necessary for theactivation of the reverse transcriptase are chelated, the reversetranscriptase may lose its activity. Therefore, it is suitable to use achelating agent of comparatively weak chelating power.

Examples of such a chelating agent of comparatively weak chelating powerinclude deoxynucleotide triphosphates (dNTPs). The chelating agent ofcomparatively weak chelating power is suitably used in an approximatelyequimolar amount of the metal ion. When a deoxynucleotide triphosphateis used as the chelating agent, for example, it is suitable to add anapproximately equimolar amount of deoxynucleotide triphosphate as to themetal ion.

Accordingly, the amount of the chelating agent can be suitably decidedwith consideration to the chelating power as to the objective metal ion,so that the reverse transcriptase activity can be maintained and thefragmentation of mRNAs can be prevented. The deoxynucleotidetriphosphates, DATP, dGTP, dCTP and dTTP, may be used alone or in anycombination thereof. All of the four kinds of dNTPs, DATP, dGTP, dCTPand dTTP, may be used together. Since these can serve also as substratesof the reverse transcription, all of them are usually used together.

The solution containing RNAs can further contain one or morepolyalcohols such as glycerol.

According to the fifth embodiment of the present invention, heatstability of RNAs is improved even though the an RNA containing solutionfurther contains metal ions such as magnesium ions or manganese ionsand/or tris(hydroxymethyl)aminomethane. In addition, the aboveimprovement is obtainable, for example, at a temperature of 40-100° C.,preferably 45-90° C.

EXAMPLES

The present invention will be further explained in detail with referenceto the following examples.

Example 1

Stability of mRNA in metal ion-containing buffer optionally containingdNTP

To examine stability of RNAs in a buffer (50 mM Tris, pH 8.3, 3 mMMgCl₂) containing several additives, total River RNAs were incubated invarious buffer solutions of the compositions listed below.

TABLE 1 Lane 1 50 mM Tris, pH 8.3, 3 mM MgCl₂, 15% (v/v) glycerol 2 50mM Tris, pH 8.3, 3 mM MgCl₂ 3 50 mM Tris, pH 8.3, 3 mM MgCl₂, 2 mM dNTP4 50 mM Tris, pH 8.3, 3 mM MgCl₂, 3 mM dNTP 5 50 mM Tris, pH 8.3, 3 mMMgCl₂, 4 mM dNTP 6 50 mM Tris, pH 8.3, 3 mM MgCl₂, 3 mM dNTP, 15%glycerol 7 Sterilized water

To visualize fragmentation of RNAs after the incubation, the sampleswere subjected to agarose gel electrophoresis as described by Sambrook(Molecular Cloning, The second edition pp. 7.43-7.45). The gel wasstained with ethidium bromide and the degree of the RNA fragmentationwas evaluated by comparing relative band intensities of rRNA. Theresults of the agarose gel electrophoresis are shown in FIG. 1 (Lanes1-7).

As shown in Lane 1, the RNAs were not sufficiently protected from thefragmentation by glycerol in the presence of magnesium ion (free Mg²⁺)of high concentration, i.e., when incubated in 50 mM Tris, pH 8.3, 3 mMMgCl₂, 15% (v/v) glycerol. In fact, the degree of the fragmentation wassimilar to that obtained in 50 mM Tris, pH 8.3, 3 mM MgCl₂ in theabsence of glycerol (Lane 2).

As shown in Lane 3, the fragmentation of RNA was not prevented yet bytreatment with 50 mM Tris, pH 8.3, 3 mM MgCl₂, 2 mM dNTP.

On the other hand, the fragmentation of RNA was partially prevented inthe condition of 50 mM Tris, pH 8.3, 3 mM MgCl₂, 3 mM dNTP (same molarconcentrations of Mg²⁺and NTP) as shown in Lane 4.

Further, as shown in Lane 5, in 50 mM Tris, pH 8.3, 3 mM MgCl₂, 4 mMdNTP, i.e., in a condition that the concentration of NTP was higher thanthat of Mg²⁺by 1 mM, the RNAs were very stable. However, it was alsofound that the activity of the reverse transcriptase is reduced underthis condition.

So, 15% glycerol was added to 50 mM Tris, pH 8.3, 3 mM MgCl₂, 3 mM dNTP(same molar concentrations of NTP and Mg²⁺) and the RNAs did not undergofragmentation under this condition as shown in Lane 6. It was also foundin a separate experiment that the activity of reverse transcriptase wascompletely maintained under this condition.

Under the condition of Lane 6, stability of the RNAs was almost similarto that obtained in Lane 7, i.e., in sterilized water.

Example 2

Improvement of reverse transcription efficiency by making reversetranscriptase heat-resistant

To examine reverse transcription activity under the novel condition ofLane 6, cDNAs were synthesized using RNAs as template. The RNAs weretranscribed in vitro by T7 RNA polymerase as mentioned below. The RNAswere prepared by transcribing pBluescript II SK, which had been cleavedinto a linear form with a restriction enzyme NotI, in vitro with T7 RNApolymerase. This reaction was initiated from T7 promoter described inthe instruction of pBluescript II SK.

The resulting products were evaluated. By using RNAs as a templatetranscribed in vitro and evaluating the products by electrophoresis,reverse transcription efficiencies of the samples can be compared withone another and thereby non-specific transcription termination whichleads to premature termination of reverse transcription and/or reductionof reaction efficiency can be evaluated.

As a control, the following standard buffer condition was used: 50 mMTris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 0.75 mMeach of dNTPs (DATP, dGTP, dCTP and dTTP).

In the above standard buffer condition, 1 μg of template RNA, 400 ng ofprimer (20mer SK primer, CGCTCTAGAACTAGTGGATC) and 200 units ofSuperscript II were prepared and the final volume was adjusted to 20 μl.0.2 μl of [α-³²P]dGTP was used for labeling of reverse transcriptionproducts. The RNA and the primer were incubated at 65° C. before theother substrates were added. Then, the reaction was performed at 42° C.for 1 hour. The reaction products were subjected to denaturing agaroseelectrophoresis and electrophoretic patterns were examined byautoradiography to evaluate recoveries of full length cDNAs and rates ofshort products obtained from incomplete elongation. The results areshown in Lane 1 of FIG. 2.

The reverse transcriptase Superscript II was inactivated at atemperature of 50° C. in the above standard buffer condition.

The following buffer condition for reverse transcription was used toverify that addition of oligosaccharide stabilizes the enzyme reaction:50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol,each 0.75 mM of dNTPs (DATP, dGTP, dCTP, dTTP), 20% (w/v) trehalose and20% (v/v) glycerol.

1 μg of template RNA, 400 ng of primer (20mer SK primer) and 200 unitsof Superscript II were reacted in 24 μl of aqueous solution under theabove buffer condition. 0.2 μl of [α-³²P]dGTP was used for labeling ofreverse transcription products. Under this condition, the reversetranscriptase Superscript II exhibited higher activity than the controlreaction at a normal temperature (42° C.). The primer and the templateRNAs were annealed at 37° C. for 2 minutes and the enzyme activity wasmeasured at 60° C.

The reaction products were subjected to denatured agaroseelectrophoresis as described above, and electrophoretic patterns wereexamined by autoradiography to evaluate recoveries of full length cDNAsand rates of short products obtained from incomplete elongation. Theresults are shown in FIG. 2.

As shown in Lane 1, products resulted from premature termination ofreverse transcription at specific sites or non-specific termination ofreverse transcription were seen under the standard buffer condition at42° C.

As shown in Lane 2, at 42° C. as in Lane 1, such products resulted frompremature termination as mentioned above were also observed even though20% trehalose and 20% glycerol were added.

As shown in Lane 3, when the temperature was raised to 60° C., theamount of products obtained from prematurely terminated synthesis becamevery small and full length products were synthesized.

As shown in Lane 5, when 0.125 μg/μl of BSA was added to the conditionof Lane 3, the enzyme activity was further stabilized. However, BSAalone without 20% trehalose and 20% glycerol did not make the enzymesufficiently heat-resistant.

As shown in Lane 4, when 0.05% of Triton X100 was added to the conditionof Lane 3, the amount of incomplete reverse transcription products wasfurther reduced. However, the whole activity of the reversetranscriptase was slightly reduced.

When the reaction was performed under the same condition as Lane 3except that glucose or maltose was used instead of trehalose, theelectrophoretic pattern showed again that the amount of productsobtained from prematurely terminated synthesis became very small andfull length products were synthesized.

Synthesis of CDNA from mRNA template

From the findings in the above Examples 1 and 2, it became clear thatcDNAs could be synthesized with high efficiency starting from mRNAs byusing the buffer condition of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 MMMgCl₂, 10 mM dithiothreitol, 0.75 mM each of dNTPs, 20% (w/v) trehaloseand 20% (v/v) glycerol. The reaction conditions were as follows: 1 μg oftemplate RNA, 400 ng of oligo-dT(12-18) primer and 200 units ofSuperscript II were reacted in a volume of 24 μl in the presence of[α-³²P]dGTP, the primer and the template RNAs were annealed at 37° C.for 2 minutes and the enzyme activity was measured at 60° C.

The obtained first strand cDNA chains are used in long RT-PCR or inconstruction of full length cDNA libraries.

Example 3

Reaction was performed under the same condition as Lane 3 of Example 2except that arabitol, sorbitol, levulose, xylitol or betain was usedinstead of trehalose. The electrophoretic pattern showed again that theamount of products obtained from prematurely terminated synthesis becamevery small and full length products were synthesized as in Lane 3 ofExample 1.

What is claimed is:
 1. A method for improving heat stability of RNAs ina solution wherein the solution comprises a polyalcohol, magnesium ions,and one or more chelation agents for the magnesium ions, and whereinsaid chelating agents are equimolar with or in excess to the magnesiumions.
 2. The method of claim 1, wherein the chelating agent comprisesone or more deoxynucleotide triphosphates.
 3. The method of claim 1,wherein the magnesium ions are added as MgCl₂.
 4. The method of claim 1,wherein the heat stability is improved at a temperature of 40-100° C. 5.The method of claim 1, wherein the solution further contains more thanone polyalcohol.
 6. The method of claim 1, wherein the deoxynucleotidetriphosphates are dATP, dCTP, dGTP, and dTTP.